Mitochondrial DNA (mtDNA) is known to show strict maternal inheritance in mammals. We have previously found that sperm mtDNA was eliminated from the cytoplasm of embryonic cells by the 2-cell-stage in early embryogenesis, using highly sensitive PCR methods (Kaneda, H. et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4542–4546; and Shitara, H. et al., (1998) Genetics 148, 851–857). This phenomenon could cause the maternal inheritance of mtDNA.
To observe directly and visually the intracellular behavior of sperm mitochondria during early embryogenesis, mitochondrial fraction, cells and tissue have been stained by Rhodamine 123 or by MitoTracker (Molecular Probes, OR; cat. M7512) and observed under a fluorescence microscope (Kaneda, H. et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4542–4546; Cummins, J. M. et al., (1997) Zygote 5, 301–308; Sutovsky, P. et al., (1996) Biol. Reprod. 55, 1195–1205; and Sutovsky, P. et al., (2000) Biol. Reprod. 63, 582–590). Using these fluorescent dyes, mitochondria can be clearly and specifically stained in the middle piece of a sperm cell.
However, several drawbacks are known regarding the above methods. The above dyes are easily bleached under the irradiation of UV light, and even without irradiation of UV light the dyes rapidly become bleached. The procedures involved in the staining such as centrifugation and resuspension damaged cells and mitochondria, and consequently the fertility of sperm is remarkably reduced, and the dyes are easily released after staining. These drawbacks make it difficult to conduct sequential or repeated observations of mitochondria.
Instead of vital staining dyes, the use of green fluorescent protein (GFP), which has been widely applied as a noninvasive chemiluminescent reporter molecule, enables the visualization of the localization and/or migration of proteins of interest in intracellular compartmentation. GFP can also be used for real-time visualization of intracellular organelles. In particular, Rizzuto et al. (Rizzuto, R. et al., (1995) Curr. Biol. 5, 635–642; and Rizzuto, R. et al., (1996) Curr. Biol. 6, 183–188) reported that GFP was accumulated exclusively in the mitochondria of HeLa cells by transfection of a GFP cDNA clone linked with a polynucleotide encoding the N-terminus of the mitochondrial cytochrome c oxidase subunit VIII precursor protein (mtGFP). Fluorescence was observed as a rod-like shape typical of mitochondria, suggesting that GFP is accumulated in mitochondria. Moreover, the reduction of GFP fluorescence was not at all or less observed by repeated UV-irradiation, whereas the fluorescence of rhodamine 123, a vital staining dye specific to mitochondria, was dramatically reduced under the same conditions.
As described above, it has been expected that GFP linked with an importing signal to a certain organelle has wide applications in real-time observation of the intracellular organelle.
However, the above methods have drawbacks in that applicable cells are limited to cells such as established cultured cells and cells of tissue that can be subjected to primary culture, and that the behavior of mitochondria during ontogenesis and the dynamics of mitochondria in lesions during pathological development cannot be observed.
To express mitochondria-localized GFP in every tissue in an individual animal, an appropriate vector must be selected. To date, expression vectors having promoter sequences to enable tissue-specific expression or vectors that enable expression in all the tissues have been invented and improved. However, there are only few expression vectors that are completely sufficient, particularly in terms of the expression level of a foreign gene.
In the meantime, apoptosis, which is also referred to as programmed cell death and by which the cell content is not released outside the cell, in contrast to necrosis by which the cell content is released extracellularly, is known to cause disorders of mitochondria involving characteristic morphological changes such as the aggregation or fragmentation of nuclei within cells and the discharge of cytochrome C. Recently, the involvement of apoptosis in ontogeny and various diseases is increasingly reported. However, most of the facts about apoptosis remained unknown.